Chromatographie et purification des diphenol oxydases du sol

Abstract

Neutral sterile and lyophilized extracts of fresh soil (NAFS Extract) degraded the following substrates: p-cresol: dl-3(3.4-dihydroxyphenylalanine)( dl DOPA): d(+) catechin and p-phenylcnediamine respectively (19.7); (5.3); (5.7) and (4.4) nmoles O 2 mg C −1 min −1 as specific activity (sp. act.). NAFS Extract was fractionated by G100 Sephadex column chromatography into two major peaks (K D ~ 0 and ~ 1) without an increase in sp. act. DEAE DE 52 cellulose chromatography separated NAFS Extract into three fractions. The first fraction was free from humic acid, relatively homogeneous on polyacrylamide gel electrophoresis and had sp. act.: dl DOPA (17.2): d(+) catechin (8.5): p-phenylenediamine (22.9). The o- and p-diphenol oxidases which accompanied this fraction were well separated as complexes on G100 Sephadex column and were liberated by dialysis against distilled water. Following isolation, we obtained an o-diphenol oxidase on dl DOPA (23.4) and a laccase activity on p-phenylenediamine (33.8) in the free state; these activities being associated with nucleoprotein. Fractions (II) and (III) appeared to be relatively homogeneous in the form of “humic acid—enzyme complexes”. Specific activity were high in fraction (III): dl DOPA (10.8): d(+) catechin (0.7); p-phenylenediamine (5.4). The diphenol oxidase activity extracted from soil (NAFS Extract) was treated by salmine and SP Sephadex C25 to remove humic matter. The EFS Extract obtained had the following sp. act.: dl DOPA (14.0); p-phenylenediamine (6.3). This EFS Extract was separated into three fractions by means of G100 Sephadex column chromatography. The Kn were (I) ~0; (II) ~ 0.52; (III) ~ 1.3 respectively. The first fraction showed an increase of sp. act. only with p-phenylenediamine (9.1) and in the following two fractions the sp. act. were not augmented. The first fraction was further fractionated by means of DEAE cellulose chromatography into four fractions: the-first and the second had no activity on dl DOPA and p-phenylenediamine. The third was an o-diphenol oxidase on dl DOPA (11.0) with traces of laccase: p-phenylenediamine (0.7). The fourth was pure laccase: p-phenylenediamine (18.1). These results suggested that electrostatic, covalent and van der Waals forces contributed to the formation of humic acid enzymes complexes, associated in the tetramer to monomer forms of diphenol oxidases.